Tumor cell-host cell interactions are critical parameters for the progression of neoplasms. Of particular importance is the relationship between neoplastic and immune cells that either mediate tumor destruction or promote tumor growth. How immune cells navigate within the tumor microenvironment, how they interact with neoplastic cells, as well as their overall contribution to the tumor micro-milieu is not well understood. The project's long-term goal is to define the cellular and molecular cues responsible for immune cell-tumor cell interaction in order to optimize immuno-therapeutic strategies. A major obstacle to such investigation has been our inability to date to image cellular interactions deep inside intact tissues at sufficiently high resolution in space and time. The objective of this proposal is to gain insight into the complex interplay between tumor and immune cells using real-time imaging at the single cell level in vivo. We propose to employ intravital two- photon microscopy (2P-IVM) to follow the activities of T cells and dendritic cells directly within their natural environment. Our hypothesis is that the migratory pattern of these immune cells within the tumor environment and the orchestration of their interactions with neoplastic cells decisively determine whether a tumor is destroyed or continues to proliferate unimpeded. We will make use of genetically engineered mouse strains having fluorescently tagged T cells or dendritic cells, whose interactions within implanted tumors we will track visually by 2P-IVM. Aim 1 will explore T cell migration within the tumor stroma, analyze T cell interactions with the extracellular matrix, and determine the kinetics of T cell-tumor cell interactions. Endogenous T cells that arise in response to tumor challenge will be compared to adoptively transferred, tumor associated antigen-specific T cells generated in vitro. Aim 2 will follow the migratory behavior of dendritic cell subsets within tumors, determine whether dendritic cells interact with tumor cells, and examine whether they establish contacts with T cells. These experiments will improve our understanding of the events leading to tumor cell destruction and/or the induction of immunologic tolerance to tumor antigens. The implication of this research is that an in-depth understanding of immune cell-tumor cell communication will, in the long-term, help to generate improved cellular immuno-therapeutic approaches.